Nov 12, 2020

Public workspacelow input ChIP-sequencing of immune cells

DOI
dx.doi.org/10.17504/protocols.io.bja3kign
  • 1Institute of Immunology & Infection Research, University of Edinburgh, Edinburgh, United Kingdom
Wiebke Nahrendorf
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DOI: dx.doi.org/10.17504/protocols.io.bja3kign
External link: https://doi.org/10.7554/eLife.63838
Protocol Citation: Wiebke Nahrendorf, Philip J Spence 2020. low input ChIP-sequencing of immune cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bja3kign
Manuscript citation:
Wiebke Nahrendorf, Alasdair Ivens and Philip J Spence Inducible mechanisms of disease tolerance provide an alternative strategy of acquired immunity to malaria. biorxiv 2020 doi.org/10.1101/2020.10.01.322180
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
we developed this low input ChIPseq protocol to look at genome-wide epigenetic changes in histone modifications. we start with just 60,000 immune cells isolated directly ex vivo by flow sorting from mouse spleens and bone marrow. for examples of the quality of the generated data see Figure 6 and S8 of our publication: www.biorxiv.org/content/10.1101/2020.10.01.322180v1. all ChIPseq data is publicly available (GEO accession number GSE150478).
Created: August 03, 2020
Last Modified: November 12, 2020
Protocol Integer ID: 39995
Keywords: ChIPseq, histone modification, epigenetic reprogramming, low input,
Abstract
Cells can stably (and heritably) alter their gene expression profile through epigenetic modifications. Histones package DNA into chromatin and can be post-translationally modified - most prominently by methylation and acetylation. These histone modifications alter chromatin structure and DNA accessibility. We optimised a protocol for reliable high quality chromatin immunoprecipitation followed by DNA sequencing (ChIPseq) starting with just 60,000 monocytes isolated directly from mouse tissues by flow sorting. Our protocol can easily be adapted to other mouse or human cell types to interrogate the genome-wide distribution of histone modifications or transcription factor binding sites in immune cells directly ex vivo.
Image Attribution
icons credit: the noun project - thenounproject.com
Materials
MATERIALS
ReagentChloroform-Isoamyl Alcohol Sigma AldrichCatalog #25666
Reagent20X EvaGreenBiotiumCatalog #31000
ReagentAgencourt AMPure XP magnetic beads Beckman CoulterCatalog #A63880
ReagentAgilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
ReagentcOmplete ULTRA Tablets, Mini, EDTA-free, EASYpackRocheCatalog #05 892 791 001
ReagentSodium Butyrate 500 mg Stemcell TechnologiesCatalog #72242
ReagentGlycineSigmaCatalog #50046
ReagentQubit® Assay TubesLife TechnologiesCatalog #Q32856
ReagentDynaMag™-2 Magnet Life TechnologiesCatalog #12321D
Reagent4% Paraformaldehyde in PBSAlfa AesarCatalog #J61899-AK
ReagentDNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
ReagentUltraPure™ DEPC-treated WaterThermo FisherCatalog #10813012
ReagentIMDMThermo FisherCatalog #12440053
ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)Thermo FisherCatalog #15593049
ReagentPBS, pH 7.2Thermo FisherCatalog #20012019
ReagentRNase Cocktail™ Enzyme MixThermo FisherCatalog #AM2286
ReagentDNAZap™ PCR DNA Degradation SolutionsThermo FisherCatalog #AM9890
ReagentQubit™ dsDNA HS Assay KitThermo FisherCatalog #Q32851
ReagentEppendorf Safe-Lock Tubes 1.5 mL PCR clean colorless 500 tubesEppendorfCatalog #022363212
ReagentTrue MicroChIP kitDiagenodeCatalog #C01010130
Reagent1.5 ml Bioruptor Pico Microtubes with CapsDiagenodeCatalog #C30010016
ReagentCorning 15mL PP Centrifuge Tubes with CentriStar Cap Sterile CorningCatalog #430791
ReagentFalcon® 5 mL Round Bottom High Clarity PP Test Tube with Snap Cap SterileCorningCatalog #352063
ReagentDrosophila spike-in chromatinActive MotifCatalog #61686
Reagentspike-in antibodyActive MotifCatalog #61686
ReagentH3K27ac Antibody - ChIP-seq GradeDiagenodeCatalog #C15410196
ReagentH3K4me1 Antibody - ChIP-seq GradeDiagenodeCatalog #C15410037-50
ReagentH3K9me3 Antibody - ChIP-seq GradeDiagenodeCatalog #C15410193
ReagentDiaMag protein A-coated magnetic beads (ChIP-seq grade)DiagenodeCatalog #C03010020
ReagentMicroPlex Library Preparation Kit v2 (12 indexes)DiagenodeCatalog #C05010012
ReagentMicroChIP DiaPure columnsDiagenodeCatalog #C03040001
ReagentLightcycler 480 multiwell plate 96 clearRocheCatalog #05102413001
ReagentMicroAmp Optical 8-Cap Strip lidsThermo FisherCatalog #4323032
ReagentTE Buffer Tris-EDTA 1X Solution pH 8.0Fisher ScientificCatalog #10224683
ReagentPremium Fetal Bovine Serum (FBS)Thermo FisherCatalog #16000044
ReagentHBSS no calcium no magnesium no phenol redThermo FisherCatalog #14175053
ReagentCertified Molecular Biology AgaroseBIO-RADCatalog #1613100
FLOW BUFFER
PBS
2 % filtered (0.22 μm) heat-inactivated FBS
5 mM EDTA

> can be stored for 2 weeks at Temperature4 °C

FIXATION BUFFER –1 ml per 1x107cells
PBS
10 % filtered (0.22 μm) heat-inactivated FBS
1 % Paraformaldehyde

> make fresh just before use, equilibrate to TemperatureRoom temperature before use

QUENCH BUFFER – use at final concentration of 125 mM Glycine
2.5 M Glycine in PBS (1.87 g in 10 ml PBS; gently heat to 37 °C in water bath, then rotate for Duration03:00:00 at TemperatureRoom temperature to dissolve completely)

> can be stored for 2 weeks at Temperature4 °C , equilibrate to TemperatureRoom temperature before use

COLLECTION MEDIA
IMDM (specifically formulated for mouse cells - if working with human cells use RPMI instead)
5 % filtered (0.22 μm) heat-inactivated FBS

HBSS + PIC (protease inhibitor cocktail)
1 complete ULTRA Protease Inhibitor Cocktail tablet per 10 ml HBSS, rotate for 3 h at TemperatureRoom temperature to dissolve completely

> store at Temperature4 °C for no longer than 1 week

100 mM sodium butyrate – use at final concentration of 5 mM sodium butyrate
dissolve in DEPC water

> store aliquots (single use – do not freeze/thaw) at Temperature-20 °C for no longer than 3 months

Drosophila spike in chromatin
stock: 10 ng/ μl
dilute in DEPC water to 40 pg/ μl

> store aliquots (single use – do not freeze/thaw) at Temperature-80 °C for no longer than 6 months

Protocol materials
ReagentFalcon® 5 mL Round Bottom High Clarity PP Test Tube with Snap Cap SterileCorningCatalog #352063
ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)Thermo FisherCatalog #15593049
ReagentDNAZap™ PCR DNA Degradation SolutionsThermo FisherCatalog #AM9890
ReagentAgilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
ReagentQubit® Assay TubesLife TechnologiesCatalog #Q32856
ReagentDNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
ReagentRNase Cocktail™ Enzyme MixThermo FisherCatalog #AM2286
ReagentTrue MicroChIP kitDiagenodeCatalog #C01010130
ReagentH3K4me1 Antibody - ChIP-seq GradeDiagenodeCatalog #C15410037-50
ReagentMicroChIP DiaPure columnsDiagenodeCatalog #C03040001
ReagentHBSS no calcium no magnesium no phenol redThermo FisherCatalog #14175053
ReagentcOmplete ULTRA Tablets, Mini, EDTA-free, EASYpackRocheCatalog #05 892 791 001
ReagentGlycineMerck MilliporeSigma (Sigma-Aldrich)Catalog #50046
Reagent20X EvaGreenBiotiumCatalog #31000
ReagentQubit™ dsDNA HS Assay KitThermo FisherCatalog #Q32851
Reagent4% Paraformaldehyde in PBSAlfa AesarCatalog #J61899-AK
ReagentIMDMThermo FisherCatalog #12440053
ReagentLightcycler 480 multiwell plate 96 clearRocheCatalog #05102413001
ReagentSodium Butyrate 500 mg STEMCELL Technologies Inc.Catalog #72242
ReagentMicroAmp Optical 8-Cap Strip lidsThermo FisherCatalog #4323032
ReagentTE Buffer Tris-EDTA 1X Solution pH 8.0Fisher ScientificCatalog #10224683
ReagentH3K27ac Antibody - ChIP-seq GradeDiagenodeCatalog #C15410196
ReagentCertified Molecular Biology AgaroseBio-Rad LaboratoriesCatalog #1613100
ReagentChloroform-Isoamyl Alcohol Merck MilliporeSigma (Sigma-Aldrich)Catalog #25666
ReagentEppendorf Safe-Lock Tubes 1.5 mL PCR clean colorless 500 tubesEppendorfCatalog #022363212
Reagent1.5 ml Bioruptor Pico Microtubes with CapsDiagenodeCatalog #C30010016
ReagentDrosophila spike-in chromatinActive MotifCatalog #61686
Reagentspike-in antibodyActive MotifCatalog #61686
ReagentDiaMag protein A-coated magnetic beads (ChIP-seq grade)DiagenodeCatalog #C03010020
ReagentDynaMag™-2 Magnet Life TechnologiesCatalog #12321D
ReagentUltraPure™ DEPC-treated WaterThermo FisherCatalog #10813012
ReagentPBS, pH 7.2Thermo FisherCatalog #20012019
ReagentH3K9me3 Antibody - ChIP-seq GradeDiagenodeCatalog #C15410193
ReagentMicroPlex Library Preparation Kit v2 (12 indexes)DiagenodeCatalog #C05010012
ReagentPremium Fetal Bovine Serum (FBS)Thermo FisherCatalog #16000044
ReagentAgencourt AMPure XP magnetic beads Beckman CoulterCatalog #A63880
ReagentCorning 15mL PP Centrifuge Tubes with CentriStar Cap Sterile CorningCatalog #430791
ReagentTrue MicroChIP kitDiagenodeCatalog #C01010130
ReagentDynaMag™-2 Magnet Life TechnologiesCatalog #12321D
ReagentMicroChIP DiaPure columnsDiagenodeCatalog #C03040001
ReagentMicroPlex Library Preparation Kit v2 (12 indexes)DiagenodeCatalog #C05010012
ReagentQubit™ dsDNA HS Assay KitThermo FisherCatalog #Q32851
ReagentAgilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
ReagentAgencourt AMPure XP magnetic beads Beckman CoulterCatalog #A63880
DNA-Protein crosslinking and cell sorting
DNA-Protein crosslinking and cell sorting
1d
1d
prepare single cell suspensions and lyse erythrocytes at TemperatureRoom temperature

RECOMMENDATION: work in 15 ml sterile, RNAse/ DNAse free, non pyrogenic polypropylene conical tubes until step 11.

NOTE: this protocol was optimised to isolate monocytes from mouse spleens and bone marrow - but it can be easily adapted to work with most other mouse and human tissues and cell types. adhere to best practice for your tissue when preparing single cells suspensions and lysing erythrocytes.
count cells, then Fc block and antibody stain in FLOW BUFFER* (scale appropriately: stain max 2x107 cells in Amount1 mL ) for Duration00:20:00 at TemperatureRoom temperature

* Buffers and Solutions in bold capitals are described in detail in Materials section

NOTE: design, titrate and test your antibody panel carefully beforehand.
wash cells twice in PBS Centrifigation350 x g, Room temperature, 00:05:00
gently resuspend cells in FIXATION BUFFER* (Amount1 mL for every 1x107 cells)

CRITICAL: warm FIXATION BUFFER to TemperatureRoom temperature before use.
incubate for exactly Duration00:10:00 at TemperatureRoom temperature , gently flick to mix occasionally

add TemperatureRoom temperature QUENCH BUFFER (final Glycine concentration Concentration125 millimolar (mM) : for every Amount1 mL FIXATION BUFFER added in step 4 add Amount50 µL QUENCH BUFFER)

incubate for Duration00:05:00 at TemperatureRoom temperature , gently flick to mix occasionally
Centrifigation450 x g, 4°C, 00:10:00 , slow brake

NOTE: faster, longer centrifugations going forward, since cell velocity changes after fixation.
aspirate supernatant carefully (leave approx. Amount100 µL ) and resuspend cells in Amount12 mL cold PBS
Centrifigation450 x g, 4°C, 00:10:00 , slow brake
aspirate supernatant carefully (leave approx. Amount100 µL ) and resuspend cells in Amount3 mL cold FLOW BUFFER
SORT 60,000 desired cells on BD FACS Aria III or similar cell sorter (85 um nozzle, sort precision mode: purity, sample and collection chamber Temperature4 °C ) into 5 ml polypropylene FACS tubes with Amount2.5 mL COLLECTION BUFFER.

RECOMMENDATION: sort several technical replicates from one biological sample and chromatin-immunoprecipitate each replicate with an antibody against a different histone modifications. in this way you will get a more detailed picture of the epigenetic landscape within each biological sample.

NOTE: always perform a test sort beforehand, where you set up all parameters, gates and compensation ready for your big ChIPseq sort day. check cell recovery - some cell types are very fragile and may require alteration of sort or collection parameters for optimal viability. our recovery was 40 - 60 % of sorted cells i.e. we continue the protocol with approx 30,000 cells. always check the purity of your sort before and after your last sample (and in between if you encountered any problems): > 95% of sorted cells should fall in the gates for your population of interest and debris should be minimal.
Critical
Centrifigation450 x g, 4°C, 00:10:00 , slow acceleration, slow brake f
aspirate supernatant carefully (leave approx Amount50 µL behind)
resuspend in Amount2 mL cold HBSS + protease inhibitors (PIC) + Concentration5 millimolar (mM) sodium butyrate
Centrifigation450 x g, 4°C, 00:10:00 , slow acceleration, slow brake
aspirate supernatant carefully (leave approx Amount50 µL behind)
flash freeze cell pellet in methanol bath on dry ice and store at Temperature-80 °C for up to 3 months
Pause
cell lysis and chromatin shearing
cell lysis and chromatin shearing
4h
4h
the following section uses reagents from the True MicroChIP kit (Diagenode, #C01010130) with a modified protocol

NOTE: work in area designated for low input DNA work, use designated pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNAZap before starting.

ReagentTrue MicroChIP kitDiagenodeCatalog #C01010130
Download TrueMicroChIP-kit-manual.pdfTrueMicroChIP-kit-manual.pdf

equilibrate lysis buffer tL1 to TemperatureRoom temperature (all crystals should be dissolved) and add protease inhibitor cocktail (PIC, from True MicroChIP Kit)
for 1 sample: Amount25 µL tL1 + Amount0.125 µL PIC (1:200)
thaw samples slowly TemperatureOn ice and add Amount1 mL ice-cold HBSS + PIC
Centrifigation450 x g, 4°C, 00:10:00 , slow acceleration, slow brake
aspirate supernatant carefully (leave as little behind as possible), keep pellets TemperatureOn ice
add Amount25 µL tL1 with PIC to the cell pellet – gently vortex to resuspend and flick until bubbles form
incubate for Duration00:05:00 TemperatureOn ice
add Amount75 µL HBSS with PIC, mix by pipetting and transfer to 1.5 ml Bioruptor Pico microtubes


using the Bioruptor Pico sonicate for 5 cycles 30 sec ON 30 sec OFF to shear the chromatin.
Equipment
Bioruptor Pico sonication device
NAME
Sonicator
TYPE
Diagenode
BRAND
B01060010
SKU
NOTE: sonication time and intervals are unique for each cell type. for optimal ChIPseq chromatin should be sheared into 100 - 300 bp fragments. see [QC chromatin shearing] in step 30.1 for how to optimise shearing.
Critical
briefly vortex and place TemperatureOn ice
Centrifigation14000 x g, 4°C, 00:10:00
transfer supernatant (=Amount100 µL sheared chromatin) to 1.5 ml DNA LoBind tube
sheared chromatin can be stored at Temperature-80 °C for up to 8 weeks or immediately immunoprecipitated (see next section)

Pause
OPTIONAL: optimise Chromatin shearing for cell type of interest [uses one flow-sorted technical replicate]

use reagents from the True MicroChIP kit (Diagenode, #C01010130) with a modified protocol. during optimisation it may pay off to use the designated Chromatin shearing optimization kit – high SDS (Diagenode, #C01020012)
  1. start with Amount100 µL sheared chromatin in 1.5 ml DNA LoBind tube
  2. dilute RNase cocktail (Amount1 µL + Amount150 µL DEPC-treated water) and add Amount2 µL to the sheared chromatin
  3. incubate Duration01:00:00 at Temperature37 °C
  4. add Amount100 µL elution buffer tE1 and Amount8 µL elution buffer tE2, mix thoroughly by pipetting
  5. decrosslink proteins from DNA for at least Duration04:00:00 or DurationOvernight in a ThermoMixer (1300 rpm) at Temperature65 °C
  6. spin tubes briefly
  7. add Amount200 µL TemperatureRoom temperature Phenol/Chloroform/Isoamyl alcohol 25:24:1
  8. vortex for Duration00:00:15 , incubate for Duration00:10:00 on rotating wheel at TemperatureRoom temperature
  9. Centrifigation14000 x g, Room temperature, 00:02:00
  10. transfer aqueous phase to new 1.5 ml DNA LoBind tube
  11. add Amount200 µL TemperatureRoom temperature Chloroform/Isoamyl alcohol 24:1
  12. vortex for Duration00:00:15 , incubate for Duration00:10:00 on rotating wheel at TemperatureRoom temperature
  13. Centrifigation14000 x g, Room temperature, 00:02:00
  14. transfer aqueous phase to new 1.5 ml DNA LoBind tube ( approx Amount150 µL )
  15. to precipitate the DNA add:
Amount15 µL tP1
Amount2 µL tCP1
Amount2 µL tCP2
Amount1 µL ice cold 100 % Ethanol
16. incubate at Temperature-80 °C for Duration00:30:00
17. Centrifigation14000 x g, 4°C, 00:25:00
18. carefully discard supernatant and add Amount500 µL ice cold 70 % Ethanol
19. Centrifigation14000 x g, 4°C, 00:10:00
20. carefully remove all supernatant and allow pellet to air-dry for max. Duration00:05:00
21. resuspend pellet in Amount12 µL TE: to assess DNA fragment size distribution and integrity use Amount1 µL for Bioanalyzer HS DNA Chip (see step 113 for details) and Amount10 µL to run on a 1.5 % Agarose gel at 100 V for Duration01:00:00 (use a 100 kb ladder)
22. stain and assess gel image
Expected result
sheared chromatin fragments should be between 100 and 300 bp


8h
Optional
chromatin immunoprecipitation (ChIP)
chromatin immunoprecipitation (ChIP)
1d
1d
the following section uses reagents from the True MicroChIP kit with a modified protocol

NOTE: work in area designated for low input DNA work, use designated pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNAZap before starting.
add protease inhibitor cocktail (True MicroChIP Kit) to Chip buffer tC1
for 1 sample: Amount100 µL tC1 + Amount0.5 µL PIC
add Amount100 µL tC1 + PIC to Amount100 µL sheared chromatin
OPTIONAL: to normalise for technical variation between samples from this point onwards spike in a small amount of Drosophila melanogaster chromatin into each sample. an antibody against the Drosophila-specific histone variant H2Av then reliably pulls down a fraction of the Drosophila chromatin. this should happen consistently across all samples. after sequencing, the ratio of data mapping to the Drosophila genome vs your organisms genome creates a normalisation factor for each sample. you can then normalise your experimental tag counts by this factor. for more information: https://www.activemotif.com/catalog/1091/chip-normalization

add Amount140 pg Drosophila spike-in chromatin
(if you do this also add Amount0.3 µg spike-in antibody in step 36)
CITATION
Egan B, Yuan CC, Craske ML, Labhart P, Guler GD, Arnott D, Maile TM, Busby J, Henry C, Kelly TK, Tindell CA, Jhunjhunwala S, Zhao F, Hatton C, Bryant BM, Classon M, Trojer P (2016). An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition.. PloS one.
LINK
https://doi.org/10.1371/journal.pone.0166438

Optional
CRITICAL: remove Amount10 µL (5 %) of sheared chromatin as input sample, store at Temperature4 °C in a 1.5 ml DNA LoBind tube.

NOTE: input samples (one for each biological replicate) are essential to analyse ChIPseq. it is the measurement of epigenetic landscape in your cells before immunoprecipitation - all enrichment is measured relative to it.
Critical
add your antibody of interest to the remaining 95% of sheared chromatin for immunoprecipitation:

we used antibodies against H3K27ac (Amount2 µg ), H3K4me1 (Amount5 µg ) and H3K9me3 (Amount1 µg ) to investigate activation and repression of transcription as well as the future potential to respond to stimuli.

H3K27ac marks transcription start sites to activate transcription
H3K4me1 marks enhancers and superenhancers to promote gene expression
H3K9me3 condenses DNA into heterochromatin to silence gene expression

NOTE: other ChIPseq-grade antibodies (for example against transcription factors) can be used. titrate all antibodies for optimal ChIPseq. some protocol recommend qPCR for validating titrations; however, we find that qPCR results do not predict ChIP sequencing outcome. we instead recommend a test sequencing run to validate antibodies and the concentrations they are used at.

OPTIONAL: add Amount0.3 µg spike-in antibody (see step 34)
incubate DurationOvernight on a rotating wheel (40 rpm) in the cold room Temperature4 °C

Overnight
next morning: prepare DiaMag Protein A-coated magnetic beads
for 1 sample mix Amount10 µL beads (pipette up and down > 20 times to get an even suspension) with Amount50 µL beads wash buffer tBW1 in a 1.5 ml tube
place in the DynaMag- 2 magnet and wait for Duration00:01:00
ReagentDynaMag™-2 Magnet Life TechnologiesCatalog #12321D

discard the supernatant (keep tube in magnet)
take tube out of magnet and gently resuspend the beads in Amount50 µL tBW1

place in the magnet and wait for Duration00:01:00

discard the supernatant (keep tube in magnet)
take tube out of magnet and gently resuspend the beads in Amount10 µL tBW1

remove samples from rotating wheel (keep TemperatureOn ice ) and spin briefly to collect all liquid in the bottom of the tube

add Amount10 µL of washed beads

incubate for Duration06:00:00 on a rotating wheel (40 rpm) in the cold room Temperature4 °C

washes
washes
2h
2h

the following section uses reagents from the True MicroChIP kit with a modified protocol

thorough, careful washing is key for high quality ChIPseq, since it removes non-antibody bound chromatin fragments and therefore reduces background.

NOTE: work in area designated for low input DNA work, use designated pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNAZap before starting.
place magnet TemperatureOn ice and keep samples and all buffers ice-cold throughout

remove samples from rotating wheel (keep TemperatureOn ice ) and spin briefly to collect all liquid in the bottom of the tube
place your samples in the magnet and wait for Duration00:01:00 - the beads (and the immunoprecipitated chromatin bound to them) will bind to the side of the tube facing the magnet

discard the supernatant (keep tube in magnet)
take tube out of magnet and gently resuspend the beads in Amount100 µL ice cold wash buffer tW1

NOTE: do not create bubbles.


incubate for Duration00:04:00 on a rotating wheel (40 rpm) in the cold room Temperature4 °C

remove samples from rotating wheel (keep TemperatureOn ice ) and spin briefly to collect all liquid in the bottom of the tube
place your samples in the magnet and wait for Duration00:01:00

discard the supernatant (keep tube in magnet)
take tube out of magnet and gently resuspend the beads in Amount100 µL ice cold wash buffer tW2

NOTE: do not create bubbles.


incubate for Duration00:04:00 on a rotating wheel (40 rpm) in the cold room Temperature4 °C

remove samples from rotating wheel (keep TemperatureOn ice ) and spin briefly to collect all liquid in the bottom of the tube
place your samples in the magnet and wait for Duration00:01:00

discard the supernatant (keep tube in magnet)
take tube out of magnet and gently resuspend the beads in Amount100 µL ice cold wash buffer tW3

NOTE: do not create bubbles.


incubate for Duration00:04:00 on a rotating wheel (40 rpm) in the cold room Temperature4 °C

remove samples from rotating wheel (keep TemperatureOn ice ) and spin briefly to collect all liquid in the bottom of the tube
place your samples in the magnet and wait for Duration00:01:00

discard the supernatant (keep tube in magnet)
take tube out of magnet and gently resuspend the beads in Amount100 µL ice cold wash buffer tW4

NOTE: do not create bubbles.


incubate for Duration00:04:00 on a rotating wheel (40 rpm) in the cold room Temperature4 °C

remove samples from rotating wheel (keep TemperatureOn ice ) and spin briefly to collect all liquid in the bottom of the tube


place your samples in the magnet and wait for Duration00:01:00
1m
discard the supernatant (keep tube in magnet)
DNA decrosslinking
DNA decrosslinking
18h
18h
the following section uses reagents from the True MicroChIP kit with a modified protocol

NOTE: work in area designated for low input DNA work, use designated pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNAZap before starting.

after removing wash buffer tW4 take tube out of magnet and gently resuspend the beads in Amount200 µL elution buffer tE1 (equilibrate to TemperatureRoom temperature before use - tE1 should be a clear solution)
take the input samples (10 μl) you saved in step 35 out of the fridge and add Amount190 µL elution buffer tE1
incubate both ChIP and input samples for Duration00:30:00 on a rotating wheel (40 rpm) at TemperatureRoom temperature
remove samples from rotating wheel and spin briefly to collect all liquid in the bottom of the tube
place ChIP samples in the TemperatureRoom temperature magnet and wait for Duration00:01:00

ChIP samples: keep the tube in the magnet and transfer the supernatant (= your immunoprecipitated chromatin) to a new 1.5 ml DNA LoBind tube
Critical
add Amount8 µL elution buffer tE2 to both ChIP and input samples

decrosslink proteins from DNA (for both ChIP and input samples) DurationOvernight in a ThermoMixer (1300 rpm) at Temperature65 °C

Overnight
DNA purification using Micro ChIP DiaPure columns
DNA purification using Micro ChIP DiaPure columns
1h
1h

the following section uses Micro ChIP DiaPure columns (Diagenode, #C03040001) according to manufacturers instructions

NOTE: work in area designated for low input DNA work, use designated pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNAZap before starting.

ReagentMicroChIP DiaPure columnsDiagenodeCatalog #C03040001
Download MicroChIP_DiaPure_manual.pdfMicroChIP_DiaPure_manual.pdf
spin decrosslinked samples (ChIP and input samples: 200 μl Volume) briefly to collect all liquid in the bottom of the tube
add Amount1000 µL (5 Vol) TemperatureRoom temperature ChIP DNA binding buffer and mix gently by pipetting

transfer Amount600 µL to the spin column in its collection tube

Centrifigation10000 x g, Room temperature, 00:00:30

discard the flow-through
transfer the remaining Amount600 µL to the spin column in its collection tube

Centrifigation10000 x g, Room temperature, 00:00:30

discard the flow-through
add Amount200 µL TemperatureRoom temperature DNA wash buffer

CRITICAL: make sure Ethanol was added to the buffer.

Centrifigation10000 x g, Room temperature, 00:00:30

add Amount200 µL TemperatureRoom temperature DNA wash buffer

Centrifigation10000 x g, Room temperature, 00:00:30

transfer the column to a new 1.5 ml DNA LoBind tube
to elute the DNA add Amount15.2 µL TemperatureRoom temperature DNA elution buffer directly onto the column matrix and incubate for Duration00:03:00 at TemperatureRoom temperature

Centrifigation10000 x g, Room temperature, 00:00:30 then discard the column and transfer tube with DNA TemperatureOn ice

DNA can be stored at Temperature-20 °C before library preparation for up to 2 weeks
Pause
Library preparation
Library preparation
1d
1d
the following section uses the Diagenode MicroPlex Library preparation kit v2 (Diagenode, #C05010012) according to manufacturers instructions

NOTE: work in area designated for library preperation (distinct from low input DNA area), use designated library preparation pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNA-ZAP before starting.

ReagentMicroPlex Library Preparation Kit v2 (12 indexes)DiagenodeCatalog #C05010012
Download MicroPlex-Libary-Prep-Kit-v2-manual.pdfMicroPlex-Libary-Prep-Kit-v2-manual.pdf

NOTE: keep samples and reagents TemperatureOn ice throughout.
1d
in clear 96 well Lightcycler 480 plate mix Amount10 µL ChIP-ed, purified DNA with Amount2 µL template preparation buffer and Amount1 µL template preparation enzyme

gently mix by pipetting, cap using strip lids and spin briefly to collect all liquid in the bottom of the wells
run on a standard PCR machine (settings: plate, 13 μl Volume, heated lid)
temperaturetime
22 °C25 min
55 °C20 min
4 °C∞

transfer plate back TemperatureOn ice as soon as PCR machine has cooled to Temperature4 °C , spin briefly to collect all liquid in the bottom of the wells

carefully open lids and add Amount1 µL library synthesis buffer and Amount1 µL library synthesis enzyme

gently mix by pipetting, cap using strip-lids and spin briefly to collect all liquid in the bottom of the tube
incubate once more using the same standard PCR machine (settings: plate, 15 μl Volume, heated lid)
temperaturetime
22 °C40 min
4 °C∞

transfer plate back TemperatureOn ice as soon as samples PCR machine has cooled toTemperature4 °C , spin briefly to collect all liquid in the bottom of the wells

carefully open lids and add Amount30 µL library amplification master mix:

reagentvolume/reaction
Library amplification buffer25 μl
Library amplification enzyme1 μl
EvaGreen2.5 μl
Nuclease free water1.5 μl

add Amount5 µL indexing reagent (total volume 50 μl) - to avoid cross-contamination spray index lid with DNAZap and wipe dry, change gloves after each index

NOTE: carefully consider your sequencing requirements and plan which/how many libraries you are going to pool in each lane and index samples accordingly. see below for details of the standard Illumina indices supplied with the MicroPlex Library preparation kit v2 (12 indices, Diagenode #C05010012,). a kit with 48 indices is also available: MicroPlex Library Preparation Kit v2 (48 indexes, Diagenode #C05010014).

index numberindex IDindex sequence
1iPCRtagT1ATCACGTT
2iPCRtagT2CGATGTTT
3iPCRtagT3TTAGGCAT
4iPCRtagT4TGACCACT
5iPCRtagT5ACAGTGGT
6iPCRtagT6GCCAATGT
7iPCRtagT7CAGATCTG
8iPCRtagT8ACTTGATG
9iPCRtagT9GATCAGCG
10iPCRtagT10TAGCTTGT
11iPCRtagT11GGCTACAG
12iPCRtagT12CTTGTACT


Critical
gently mix by pipetting, use sealing foil and Centrifigation1300 x g, 4°C, 00:02:00

run on real time quantitative PCR machine (Roche Lightcycler 480) to monitor library amplification
Equipment
LightCycler® 480 Instrument II
NAME
real-time quantitative PCR machine
TYPE
Roche
BRAND
05015278001
SKU

temperaturetimeramp rate
extension72 °C3 min3° C/sec
cleavage85 °C2 min3° C/sec
denaturation95 °C2 min3° C/sec
addition of indices98 °C20 sec3° C/sec
67 °C20 sec2.2 °C/sec
72 °C40 sec 3 °C/sec
repeat steps 6 to 8 four times
library amplification98 °C20 sec3 °C/sec
72 °C50 sec: record fluorescence using "single acquisition"2.2 °C/sec
repeat steps 11 & 12 for x* number of cycles
cool - hold37 °C **1 h2.2 °C/sec
* monitor fluorescence after each cycle: the optimal phase is reached when Fluorescence (465-510) linearly increases to 3.5 – 4.5. at this point stop library amplification and move to step 15 - cooling. we find it takes approx 8 - 13 cycles to amplify libraries sufficiently. the exact number of cycles will depend on how much chromatin your antibody pulls down.
** 37°C is the lowest temperature the Roche Lightcycler 480 will cool to: transfer plate containing amplified library to ice 1 min after 37°C is reached

use Amount1 µL of amplified library to quantify the amount of DNA with Qubit dsDNA HS assay Kit according to manufacturers instruction

ReagentQubit™ dsDNA HS Assay KitThermo FisherCatalog #Q32851
Equipment
Qubit Fluorometer
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
https://www.thermofisher.com/order/catalog/product/Q33238#/Q33238
LINK

Expected result
5 - 15 ng/ μl
if concentration is significantly lower return sample to real time PCR machine for extra amplification


dilute Amount1 µL of amplified library in Amount4 µL TE to asses DNA intergrity and size distribution using Bioanalyzer High Sensitivity DNA Kit according to manufacturers instructions

ReagentAgilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626

Equipment
2100 Bioanalyzer Instrument
NAME
Sizing, quantification, and sample quality control of DNA, RNA, and proteins on a single platform
TYPE
Agilent Technologies
BRAND
G2939BA
SKU

Expected result
bell curve 200 - 2000 bp, average size approx. 400 bp
Download amplified_library.pdfamplified_library.pdf


pool libraries with different indices at equal molarities in 1.5 ml DNA LoBind tube (aim to reach a volume just over Amount100 µL )
Note
molecular mass of dsDNA = 660 [g/mol/bp]

calculation:
concentration [ng/ul] * 106 * 1/660 * 1/average size [bp] = molarity [nM]


Critical
transfer library from plate to 1.5 ml DNA LoBind tube (keep TemperatureOn ice )
NOTE: libraries (individual or pooled) can be stored at Temperature-20 °C for up to 2 weeks

Pause
Library purification using AMPure beads
Library purification using AMPure beads
4h
4h
NOTE: work in area designated for library preperation (distinct from low input DNA area), use designated library preparation pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNA-ZAP before starting.
bring pooled libraries, AMPure beads and freshly prepared 80 % Ethanol to TemperatureRoom temperature
ReagentAgencourt AMPure XP magnetic beads Beckman CoulterCatalog #A63880

resuspend AMPure beads until homogenous solution and add Amount100 µL to Amount100 µL of pooled library (1:1 ratio); mix until homogenous

incubate at TemperatureRoom temperature for Duration00:05:00

spin briefly (Duration00:00:03 ) to collect all liquid in the bottom of the tube

place tube in DynaMag- 2 magnet, wait for Duration00:02:00 until all beads are bound (solution clear)
discard supernatant
add Amount300 µL 80 % Ethanol

rotate the tube clockwise by 90 °, wait for Duration00:00:10 and repeat 3 more times

10s
discard supernatant
add Amount300 µL 80 % Ethanol

rotate the tube clockwise by 90 °, wait for Duration00:00:10 and repeat 3 more times
10s
discard supernatant
spin briefly (Duration00:00:03 ) to collect all liquid in the bottom of the tube

place tube in DynaMag- 2 magnet, wait for Duration00:02:00

remove all residual Ethanol
remove tube from magnet and dry the beads with lid open for max Duration00:02:00 in ThermoMixer (Temperature37 °C )

resuspend the beads in Amount50 µL TE, spin briefly (Duration00:00:03 ) to collect all liquid in the bottom of the tube

place tube in magnet, wait for Duration00:02:00

carefully transfer the eluted DNA to a new 1.5 ml DNA LoBind tube
Critical
use Amount1 µL of pooled purified library to quantify the amount of DNA with Qubit dsDNA HS assay Kit according to manufacturers instruction (see step 112 for details)
Expected result
expect 10 ng/ μl (i.e. a total of 500 ng in 50 μl)

dilute Amount1 µL of pooled purified library in Amount4 µL TE to asses integrity and size distribution of libraries using Bioanalyzer High Sensitivity DNA Kit according to manufacturers instructions (see step 113 for details)
Expected result
bell curve 200 - 2000 bp (all small fragments removed by AMPure XP bead purification), average size approx. 400 bp
Download pooled_purified_library.pdfpooled_purified_library.pdf


pooled, purified libraries can be stored at Temperature-20 °C before sequencing for up to 2 months
Note
our samples were sequenced by Edinburgh Genomics https://genomics.ed.ac.uk on the Illumina NovaSeq S1 yielding approx 750 x 106 100 bp paired end reads per lane. we aimed for a depth of 70 x 106 paired end reads for each sample. ChIPed samples (for all different histone modifications) and matched input sample should be sequenced on the same lane. we used the motif discovery software HOMER for data anlaysis (http://homer.ucsd.edu/homer/). our ChIPseq data is publicly available: GEO accession number GSE150478.
NOTE: we subscribe to the notion that ChIPseq is qualitative (it can reveal the presence or absence of a histone modification at a particular genomic location) - not quantitative (it does not reveal biologically meaningful differences in peak height, which are often influenced by the efficiency of immunoprecipitation). please consider this when analysing your results.
CITATION
Ma Z, Wang H, Cai Y, Wang H, Niu K, Wu X, Ma H, Yang Y, Tong W, Liu F, Liu Z, Zhang Y, Liu R, Zhu ZJ, Liu N (2018). Epigenetic drift of H3K27me3 in aging links glycolysis to healthy longevity in Drosophila.. eLife.
LINK
https://doi.org/pii:e35368.10.7554/eLife.35368

CITATION
Orlando DA, Chen MW, Brown VE, Solanki S, Choi YJ, Olson ER, Fritz CC, Bradner JE, Guenther MG (2014). Quantitative ChIP-Seq normalization reveals global modulation of the epigenome.. Cell reports.
LINK
https://doi.org/10.1016/j.celrep.2014.10.018




Citations
Step 34
Egan B, Yuan CC, Craske ML, Labhart P, Guler GD, Arnott D, Maile TM, Busby J, Henry C, Kelly TK, Tindell CA, Jhunjhunwala S, Zhao F, Hatton C, Bryant BM, Classon M, Trojer P. An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition.
https://doi.org/10.1371/journal.pone.0166438